Recently, an increasing number of researchers are investigating bacterial pesticides which use an insecticidal bacteria or bacterial product as an active ingredient. For example, Bacillus thuringiensis (hereinafter, it may be referred to as B.t.) is a gram positive bacterium which is known to produce various toxins such as .alpha.-, .beta.- and .gamma.-exotoxin and .delta.-endotoxin, and is highly toxic to many pests (Angus, T. A., Nature 174: 545, 1954). Among these bacterial toxins, .delta.-endotoxin produced by B.t. during the sporulation period is at the center of interest as a crystalline inclusion protein and is currently being used practically. Various B.t. strains are known to produce crystalline proteins having properties suited as an active ingredient for pesticides, such as a narrow host spectrum which makes them highly selective, a harmlessness to animals including human beings, and environmental acceptability. The host spectrum of B.t. toxin includes many insects belonging to Lepidoptera, Diptera and Coleoptera. Bacteria of B.t. strains are classified into 5 types according to the host spectrum of the crystalline proteins produced by them.
As the result of research on the genetic engineering of such crystalline proteins, the production of insect-resistant plants transformed by a gene encoding a toxic protein has become available. For instance, Vaeck et al.( Nature 328: 33-37, 1987) (Plant Genetic Systems Inc., Belgium) succeeded in the preparation of an insect-resistant tobacco (Nicotiana) by integrating a gene encoding an insecticidal protein originated from B.t. into the tobacco genome. According to the report, the expression obtained using about the 5'-half of said gene was more efficient than that obtained using the entire gene.
Fischhoff et al. (Bio/Technology 5: 807-813, 1987, Monsanto Inc.) succeeded in the preparation of a recombinant tomato (Lycopersicon) transformed by a gene encoding an insecticidal protein of B.t. These two reports, however, failed to detect the expression product of the transformed gene by Western blotting, suggesting that a sufficient expression is hardly attained by means of naturally-occurring genes.
Regarding the translation of the gene, it is interesting that the extent of translation of a given amino acid varies between tissues. Thus, tRNAs in the albumin of corn (Trysacum), which synthesize aggressively zein, a deposited protein rich in glutamine, leucine and alanine, can participate more in the translation of genes encoding these amino acids compared to tRNAs in the embryo of corn. This means that tRNAs in a particular tissue are constructed in such a manner as to promote an optimum translation of genes encoding the desired protein such as zein which should be expressed highly in said tissue.
Wilbur et al. (Plantphysiol. 92: 1-11, 1990) teach about the difference in codon usage between bacteria and higher plants such as dicotyledonous and monocotyledonous plants. Thus, the codon usages for codons XCG and XUA are 1.8% and 3.2% in dicotyledonous plants and 6.3% and 1.4% in monocotyledonous plants. The combined codon usage for codons XXC and XXG (hereinafter, referred to as the codon XXC/G usage, wherein each of the two Xs is independently selected from the group consisting of A, G, C and T) is 45% in dicotyledon and 73.5% in monocotyledon. It is well established that GC content in genes which can be translated is higher in monocotyledon such as gramineous plants, e.g., rice plant, than in dicotyledon. As to bacterium, the codon usage varies depending on the strains. For instance, the codon usage for codons XGG, XUA, and XXC/G are 10.4%, 3.3% and 24.4%, respectively in the gene encoding .delta.-endotoxin, one of B.t. toxins produced by B.t. var kurstaki HD-1 (cryIA(b)). These facts suggest that tRNAs in plant tissues can hardly afford a sufficient translation of bacterial genes.
Recently, Prederick (Bio/Technology 8: 939-942, 1990) (Monsanto Inc.) reported for the first time that B.t. toxin can be expressed highly in cotton plant (Gossypium) transformed by a modified gene in which the 3'-half is deleted and codons are changed. Thus, a gene encoding B.t. -originated .delta.-endotoxin was chemically synthesized and ligated downstream of the 35S promoter of cauliflower mosaic virus, said promoter containing the enhancer region of the 35S promoter, and the resultant gene was used for the transformation by means of Agrobacterium. The synthetic gene was prepared from genes of Bacillus thuringiensis var. kurstaki HD-1 (cryIA(b)) and HD-73 (cryIA(c)). The synthetic gene allowed a high expression of B.t. toxin (about 0.05-0.1% of soluble protein in leaves) with an expression rate of about 50-100 times that obtained using a gene encoding native B.t. toxin. When insecticidal activity was examined by bioassay using cotton-ball worms, a protection activity on an order of about 70-100% was observed.
Frederic et al. (Proc. Natl. Acad. Sci. USA 88: 3324-3328, 1991) attempted to determine regions on a bacterial gene in which codon modifications would be effective for the expression of said gene in plants. They selected 9 regions to be modified and prepared various genes in which any or all of the 9 regions were modified, and they transformed each of them into tobacco and tomato plants using Agrobacterium. When the expression efficiency obtained by a gene which contains nine modified regions is estimated to be 100%, they found that a gene containing four modified regions within 700 bp from 5'-terminus showed about 80% ,efficiency. On the contrary, no B.t. toxin was detected in plants transformed by genes whose 3'-half region was modified. The expression efficiency was highest in the case of a gene containing a modification in the region(s) between 246 to 283 bp from the 5'-terminus, and it was 53-80% of that obtained by a gene containing nine modified regions.
Prior to the present invention, as mentioned above, Monsanto Inc. showed that a high expression of bacterial protein (B.t. protein) in plants such as cotton, tobacco, tomato and the like can be achieved through the modification of codon usages. Lubrizol Genetics Inc. disclosed a synthetic gene encoding insecticidal crystalline protein (Japanese Publication (KOKAI) No. 186989/1990), in which the codon usage is discussed. The exemplified plant, however, is merely tobacco.
As is clear from the above, prior to the present invention, expression of bacterial toxin in plants has been reported only in dicotyledonous plants. There have been no reports that show a detectable amount of insecticidal crystalline protein being expressed in monocotyledonous plants.